Many immunoassays rely on monoclonal or polyclonal antibodies (IgG) derived from mice, rabbits, goats, or sheep as recognition elements. Considerable work has demonstrated that oriented capture molecules retain more binding ability and improve the performance of the assay. However natural proteins have limited means by which they can be oriented.
With advent of molecular biology, recombinant proteins including antibody fragments are being produced and utilized in increasing numbers. These recombinant proteins can be optimized for any number of parameters including oriented immobilization, as seen in, for example, U.S. Pat. No. 7,888,134, relating to electrostatic adsorption of scFv to a negatively charged surface.
In many respects it can be even more critical to orient small recombinant antibody derivatives because they have been reduced to only the active domain, thus reducing the number of random attachment sites that will not impair function. The same is likely true of many recombinant enzymes as well.
While there have been specialized methods developed for particular surfaces or using non-natural amino acids, the vast majority of covalent protein surface immobilization as well as dye/biotin molecule attachment chemistry makes use of either a carbodiimide or succinimide ester chemistry, which links a carboxyl group on one molecule to an amine on another. The limitation for successful orientation of proteins with these chemistries is that proteins possess a number of carboxyl and amine residues spread across their surface. The challenge has been to successfully orient the protein or attach the dye/biotin in a particular location so as to preserve maximal activity of the protein.
A need exists for a facile and effective technique to control the orientation of peptides and proteins, particularly antibodies, upon their immobilization.